CD30 bispecific antibodies and method of immunotherapy of CD30+ malignancies

ABSTRACT

The present invention provides novel bispecific antibodies that bind to human CD30 and uses thereof. Methods of treating cancer using the bispecific antibodies described herein are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 62/735,576filed on Sep. 24, 2018, the contents of which is incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

N/A

BACKGROUND OF THE INVENTION

The field of the invention is novel bispecific antibodies specific tohuman CD30, and the use thereof.

Bispecific antibodies (BiAb) include antibodies or antibody-likemolecules that contain two different binding specificities by combiningtwo different binding moieties. BiAbs have been studied to be used asimmunotherapy for tumors in order to increase the immune responseagainst tumor antigens and thus tumor cells.

CD30 cell surface molecule is a member of the tumor necrosis factorreceptor (TNF-R) superfamily and a transmembrane glycoproteinpreferentially expressed by activated lymphoid cells. This family ofmolecules has variable homology among its members and includes nervegrowth factor receptor (NGFR), CD120(a), CD120(b), CD27, CD40 and CD95.Members of this family play a role in regulating proliferation anddifferentiation of lymphocytes.

CD30 was originally identified by the monoclonal antibody Ki-1, which isreactive with antigens expressed on Hodgkin and Reed-Sternberg cells ofHodgkin's disease (Schwab et al., Nature 299:65 (1982)). CD30 has beenused as a clinical marker for Hodgkin's lymphoma and relatedhematological malignancies (Froese et al., J. Immunol. 139:2081 (1987);Carde et al., Eur. J. Cancer 26:474 (1990)). It has since been found ona number of hematologic malignancies. Since the percentage ofCD30-positive cells in normal individuals is very low, CD30 in tumorcells renders it an important target for antibody mediated therapy tospecifically target therapeutic agents against CD30-positive neoplasticcells (Chaiarle, R., et al. Clin. Immunol. 90(2):157-164 (1999)).

Hodgkin Lymphoma (HL) is often treatable, with 86% surviving over 5years. However, about 30% of patients relapse, a subset of which developresistant HL. Refractory or relapsed chemo-resistant disease is morechallenging to treat: the 5-year survival rate for these patients isjust 31%. CD30 is also expressed in a substantial subset of patientswith acute myeloid leukemia (AML) which accounts for 1.2% of all cancercases in the United States and has a 5-year survival rate of just 26.6%.Relapse following initial therapy is common, and patients who relapseafter a stem cell transplantation are typically non-responsive tofurther therapeutic intervention.

Accordingly, the need exists for improved bispecific antibodies that cantarget CD30+ cancers.

SUMMARY OF THE INVENTION

The present invention addresses the aforementioned drawbacks byproviding isolated bispecific antibodies able to bind CD30 antibodiesand a T-cell or NK cell surface antigen for example CD-3. A particularembodiment is a bispecific antibody comprising an anti-CD30 and ananti-CD3 antibody or antigen binding fragment thereof covalently linked.The bispecific antibodies described herein can be used for methods oftreating patient populations having CD30+ cancers.

In one aspect, the present disclosure provides an isolated bispecificantibody capable of binding human CD30 and to a T cell surface antigencomprising a CD30 antibody or antigen binding portion thereof and a Tcell surface antigen antibody or antigen binding portion thereof,

In another aspect, the present disclosure provides an isolatedbispecific antibody capable of binding human CD30 and to a T cellsurface antigen comprising a CD30 antibody or antigen binding portionthereof and a T cell surface antigen antibody or antigen binding portionthereof, the CD30 antibody or antigen binding portion thereof comprising

(a) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:2 or a sequence with 85% similarity to SEQ ID NO:2, a CDRL2 region ofSEQ ID NO:3 or a sequence with 85% similarity to SEQ ID NO:3, and aCDRL3 region of SEQ ID NO:4 or a sequence with 85% similarity to SEQ IDNO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:6 or a sequence with 85% similarity to SEQ ID NO:6, a CDRH2 regionof SEQ ID NO:7 or a sequence with 85% similarity to SEQ ID NO:7, and aCDRH3 region of SEQ ID NO:8 or a sequence with 85% similarity to SEQ IDNO:8;

(b) a light chain variable domain comprising a CDRL1 region of SEQ IDNO: 10 or a sequence with 85% similarity to SEQ ID NO: 10, a CDRL2region of SEQ ID NO: 11 or a sequence with 85% similarity to SEQ IDNO:11, and a CDRL3 region of SEQ ID NO:12 or a sequence with 85%similarity to SEQ ID NO: 12 and a heavy chain variable domain comprisinga CDRH1 region of SEQ ID NO:14 or a sequence with 85% similarity to SEQID NO:14a CDRH2 region of SEQ ID NO:15 or a sequence with 85% similarityto SEQ ID NO:15, and a CDRH3 region of SEQ ID NO:16 or a sequence with85% similarity to SEQ ID NO:16,

(c) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:18, a CDRL2 region of SEQ ID NO:19, and a CDRL3 region of SEQ IDNO:20 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:22, a CDRH2 region of SEQ ID NO:23, and a CDRH3 region of SEQ IDNO:24,

(d) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:26, a CDRL2 region of SEQ ID NO:27, and a CDRL3 region of SEQ IDNO:28 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:30, a CDRH2 region of SEQ ID NO:31, and a CDRH3 region of SEQ IDNO:32, or

(e) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:34, a CDRL2 region of SEQ ID NO:35, and a CDRL3 region of SEQ IDNO:36 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:38, a CDRH2 region of SEQ ID NO:39, and a CDRH3 region of SEQ IDNO:40. In a preferred embodiment, the T cell surface antigen is CD3.

In another aspect, the present invention provides an isolated bispecificantibody capable of binding human CD30 and to a NK cell surface antigen,the bispecific antibody comprising a CD30 antibody or antigen bindingportion thereof and a NK cell surface antigen antibody or antigenbinding portion thereof, the CD30 antibody or antigen binding portionthereof comprising

(a) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:2 or a sequence with 85% similarity to SEQ ID NO:2, a CDRL2 region ofSEQ ID NO:3 or a sequence with 85% similarity to SEQ ID NO:3, and aCDRL3 region of SEQ ID NO:4 or a sequence with 85% similarity to SEQ IDNO:4 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:6 or a sequence with 85% similarity to SEQ ID NO:6, a CDRH2 regionof SEQ ID NO:7 or a sequence with 85% similarity to SEQ ID NO:7, and aCDRH3 region of SEQ ID NO:8 or a sequence with 85% similarity to SEQ IDNO:8;

(b) a light chain variable domain comprising a CDRL1 region of SEQ IDNO: 10 or a sequence with 85% similarity to SEQ ID NO: 10, a CDRL2region of SEQ ID NO: 11 or a sequence with 85% similarity to SEQ ID NO:11, and a CDRL3 region of SEQ ID NO: 12 or a sequence with 85%similarity to SEQ ID NO: 12 and a heavy chain variable domain comprisinga CDRH1 region of SEQ ID NO:14 or a sequence with 85% similarity to SEQID NO:14a CDRH2 region of SEQ ID NO:15 or a sequence with 85% similarityto SEQ ID NO:15, and a CDRH3 region of SEQ ID NO: 16 or a sequence with85% similarity to SEQ ID NO: 16,

(c) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:18, a CDRL2 region of SEQ ID NO:19, and a CDRL3 region of SEQ IDNO:20 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:22, a CDRH2 region of SEQ ID NO:23, and a CDRH3 region of SEQ IDNO:24,

(d) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:26, a CDRL2 region of SEQ ID NO:27, and a CDRL3 region of SEQ IDNO:28 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:30, a CDRH2 region of SEQ ID NO:31, and a CDRH3 region of SEQ IDNO:32, or

(e) a light chain variable domain comprising a CDRL1 region of SEQ IDNO:34, a CDRL2 region of SEQ ID NO:35, and a CDRL3 region of SEQ IDNO:36 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:38, a CDRH2 region of SEQ ID NO:39, and a CDRH3 region of SEQ IDNO:40. In a preferred embodiment, the NK cell surface antigen is CD16.

In yet another aspect, the present disclosure provides pharmaceuticalcompositions comprising the bispecific antibody described herein.

In another aspect, the present disclosure provides a method of treatinga patient having a CD30+ cancer, the method comprising

(a) administering a therapeutically effective amount of the bispecificantibody described herein to reduce or inhibit CD30+ cancer growth.

In another aspect, the present disclosure provides a method ofinhibiting growth of a tumor cell expressing CD30, comprising contactingthe tumor cell with an effective amount of the bispecific antibodydescribed herein such that the growth of the cell is inhibited.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere are shown, by way of illustration, preferred embodiments of theinvention. Such embodiments do not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depict the specific binding of novel CD30 antibody clones toCD30− and CD30+ cells. Flow cytometry analysis was preformed usingpurified antibody on CD30+ cell lines (SU-DHL-1, RPM16666, 562) and aCD30− cell line (OCIAML2). A commercially available α-huCD30 antibody(clone BY88) and an isotype control were also tested.

FIG. 2 depicts specific binding of the CD30 antibodies using flowcytometry on non-transduced Raji (low levels of endogenous CD30expression) and LV transduced CD30+ Raji cells (high levels of CD30expression).

FIG. 3A depicts the percent identity between the light chains of thenovel CD30 antibodies to each other and the known CD30 antibody AC10.

FIG. 3B depicts the percent identity between the heavy chains of thenovel CD30 antibodies to each other and the known CD30 antibody AC10.

FIG. 4 depicts the binding of the antibodies to CD30 as measured byELISA.

FIG. 5 depicts the percent inhibition of AC10 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 6 depicts the percent inhibition of 8D10 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 7 depicts the percent inhibition of 10C2 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 8 depicts the percent inhibition of 12B1 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 9 depicts the percent inhibition of 13H1 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 10 depicts the percent inhibition of 15B8 binding to CD30⁺ cells(SU-DHL-1 cells) by 8D10, 10C2, 12B1, 13H1, 15B8, or AC10 antibodies.

FIG. 11 is a set of bar graphs summarizing the blocking ability of theantibodies to AC10 (left) and 8D10 (right) binding to CD30⁺ cells.

FIG. 12 is a set of bar graphs summarizing the blocking ability of theantibodies to 10C2 (left) and 12B1 (right) binding to CD30⁺ cells.

FIG. 13 is a set of bar graphs summarizing the blocking ability of theantibodies to 13H1 (left) and 15B8 (right) binding to CD30⁺ cells.

FIGS. 14A-14E shows further characterization of the bispecificantibodies. FIG. 14A shows a representative drawing of the anatomy ofone embodiment of bispecific antibodies of the present invention. FIG.14B shows flow cytometry analysis of biAb binding to CD3+ or CD30+cells. FIG. 14C demonstrates the conjugation assay design. FIG. 14Dshows a summary of results from the conjugation assay. FIG. 14E shows arepresentative flow cytometry plots from the conjugation assay showing amixture of biAb-armed T cells and CD30+ tumour analysed by flow. Panel 1is unarmed T cells, panel 2 is 8D10 biAb-armed T cells, and panel 3 is10C2 biAb-armed T cells.

FIG. 14F is an example method of conjugating the CD30+ antibody and CD3antibody of the present invention.

FIG. 15 shows the expression of CD30 on tumor cells.

FIGS. 16A-16B are bar graphs depicting cytokines produced by biAb-armedT cells. 8D10 biAb or 10C2 biAb-armed T cells produce thepro-inflammatory cytokines IL-2 (FIG. 16A) and IFN-γ (FIG. 16B) whenco-cultured with CD30+ tumor cells, indicating that binding of T cellsto the biAb is sufficient to trigger T cell activation and cytotoxicity.Armed T cells cocultured with CD30− cells have much lower cytokineproduction demonstrating the specificity of this approach.

FIGS. 17A-17D demonstrate cytotoxicity of the biAbs of the presentinvention. Chromium release assay demonstrating the cytotoxicity of 8D10and 10C2 biAb-armed T cells against CD30+ tumor cells. CD30+ tumor celllines SU-DHL-1 (FIG. 17A), RPMI6666 (FIG. 17B) and Raji LV30 (FIG. 17D,Raji cells transduced with CD30 lentivirus) are killed with varyingefficacy by 8D10 and 10C2 biAb-armed T cells. Non-transduced Raji cells(FIG. 17C, CD30−) are not killed by the armed T cells. Activated T cellsmay express low levels of CD30, which could be detrimental to thesuccess of this biAb therapy.

FIGS. 18A-18B demonstrate CD30 negative control cell (FIG. 18A) are notkilled by biAb-armed T cells and that the CD30 expression on activated Tcells is low enough to avoid elimination by the 8D10 and 10C2 biAb armedT cells (FIG. 18B). Experiment was done similar to FIG. 17A-17D.

FIGS. 19A-19B demonstrates surface palsmon resonance (SPR) analysis ofCD30 mAbs using two approaches. FIG. 19A shows the results when CD30protein was immobilized on a CM5 chip, and the antibodies were flowed asan analyte to assess affinity. FIG. 19B shows the results when 8D10,10C2, or AC10 antibody was immobilized on a Protein G chip, and CD30protein was flowed as an analyte to assess affinity.

DETAILED DESCRIPTION

The present invention provides isolated bispecific antibodies that canspecifically/selectively bind to CD30+ cells and to T cells or NK cellssimultaneously. Specifically, in one embodiment, a bispecific antibodythat binds to CD30+ and the T cell surface protein CD3 is provided. Thepresent invention also provides in some embodiments methods of treatinga subject having a CD30+ tumor using the bispecific antibodies describedmore herein. In another embodiment, the present disclosure provides amethod of inhibiting growth of a tumor cell by administering aneffective amount of the bispecific antibodies described herein. In yetanother embodiment, the present disclosure provides a method ofenhancing a T cell-mediated immune response against a CD30+ tumor cell,the method comprising administering a therapeutically effective amountof the bispecific antibody described herein to increase the Tcell-mediated immune response as compared to treatment without thebispecific antibody.

The term “bispecific antibody” as used herein means an antibody, such asa recombinant antibody capable of specifically and selectivelyrecognizing and binding two different antigens. The antibody can beproduced by methods known in the art, including chemical linkage or cellfusion methods. In the present disclosure the bispecific antibody isable to bind (a) CD30 and (b) a T cell or NK cell surface antigen.Specifically, in one embodiment, the bispecific antibody is able to bind(a) CD30 and (b) CD3.

In one embodiment, the bispecific antibody comprises (a) a CD30 bindingantibody or antigen binding fragment thereof and (b) a T cell surfaceantigen biding antibody or antigen binding fragment thereof. In oneembodiment, both the CD30 and T cell surface antigen binding antibodiesor antigen binding fragments are two different antibodies that arecovalently or non-covalently linked to each other. In a specificembodiment, the bispecific antibody is a CD30 antibody covalently linkedto a CD3 antibody.

The terms “antibody” or “antibody molecule” are used hereininterchangeably and refer to immunoglobulin molecules or other moleculeswhich comprise an antigen binding domain. The term “antibody” or“antibody molecule” as used herein is thus intended to include wholeantibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies,chimeric antibodies, humanized antibodies, and antibody fragments,including single chain variable fragments (ScFv), single domainantibody, and antigen-binding fragments, genetically engineeredantibodies, among others, as long as the characteristic properties(e.g., ability to bind CD30) are retained. The term “antibody fragment”as used herein is intended to include any appropriate antibody fragmentthat displays antigen binding function, for example, Fab, Fab′, F(ab′)2,scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimersthereof and bispecific antibody fragments. Thus, the bispecificantibodies described herein may be two antibodies that are covalently ornon-covalently linked to produce a single bispecific antibody. The twoantibodies may be linked by a linker.

As stated above, the term “antibody” includes “antibody fragments” or“antibody-derived fragments” and “antigen binding fragments” whichcomprise an antigen binding domain. Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainantibodies or single chain Fv (scFv), see for instance Bird et al.,Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883(1988)). Such single chain antibodies are encompassed within the termantibody unless otherwise noted or clearly indicated by context.

Antibodies can be genetically engineered from the CDRs and monoclonalantibody sequences described herein into antibodies and antibodyfragments by using conventional techniques such as, for example,synthesis by recombinant techniques or chemical synthesis. Techniquesfor producing antibody fragments are well known and described in theart.

One may wish to engraft one or more CDRs from the monoclonal antibodiesdescribed herein into alternate scaffolds for use in the bispecificantibody. For example, standard molecular biological techniques can beused to transfer the DNA sequences encoding the antibody's CDR(s) to (1)full IgG scaffold of human or other species; (2) a scFv scaffold ofhuman or other species, or (3) other specialty vectors. If the CDR(s)have been transferred to a new scaffold all of the previousmodifications described can also be performed. For example, one couldconsult Biotechnol Genet Eng Rev, 2013, 29:175-86 for a review of usefulmethods.

The bispecific antibodies disclosed in the present invention may bemodified to be humanized antibodies which include the constant regionfrom a human germline immunoglobulin sequences. The term “recombinanthuman antibody” or “humanized antibody” includes all human antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as antibodies isolated from a host cell such as an SP2-0, NSO orCHO cell (like CHO K1) or from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes or antibodies or polypeptidesexpressed using a recombinant expression vector transfected into a hostcell. Such recombinant human antibodies have variable and in someembodiments, constant regions derived from human germline immunoglobulinsequences in a rearranged form.

For example, a humanized antibody may comprise the constant regionsderived from the human germline immunoglobulin sequence and the“framework” (FR) variable domain residues which are the variable domainresidues other than the hypervariable regions (CDRs). The framework ofthe variable domain usually consists of four FR domains (between thethree CDRs, e.g., FR1, FR2, FR3 and FR4) for both the heavy and lightchain (e.g., for light chain region would contain:FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4). Therefore, a humanized antibodymay have the constant regions and framework from a human immunoglobulinand the CDRs or hypervariable regions from the mouse monoclonalantibodies described herein.

The term “fragment” as used herein refers to fragments of biologicalrelevance (functional fragment), e.g., fragments which can contribute toor enable antigen binding, e.g., form part or all of the antigen bindingsite or can contribute to the prevention of the antigen interacting withits natural ligands. Fragments in some embodiments comprise a heavychain variable region (VH domain) and light chain variable region (VL)of the invention. In some embodiments, the fragments comprise one ormore of the heavy chain complementarity determining regions (CDRHs) ofthe antibodies or of the VH domains, and one or more of the light chaincomplementarity determining regions (CDRLs), or VL domains to form theantigen binding site. For example, a fragment is suitable for use in thepresent methods and kits if it retains its ability to bind CD30.

The term “complementarity determining regions” or “CDRs,” as usedherein, refers to part of the variable chains of immunoglobulins(antibodies) and T cell receptors, generated by B-cells and T-cellsrespectively, through which these molecules bind to their specificantigen. As the most variable parts of the molecules, CDRs are crucialto the diversity of antigen specificities generated by lymphocytes.There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively,on the amino acid sequence of a variable domain of an antigen bindingsite. Since the antigen binding sites are typically composed of twovariable domains (on two different polypeptide chains, heavy and lightchain), there are six CDRs for each antigen binding site that cancollectively come into contact with the antigen. A single whole antibodymolecule has two antigen binding sites and therefore contains twelveCDRs. Sixty CDRs can be found on a pentameric IgM molecule.

Within the variable domain, CDR1 and CDR2 may be found in the variable(V) region of a polypeptide chain, and CDR3 includes some of V, all ofdiversity (D, heavy chains only) and joining (J) regions. Since mostsequence variation associated with immunoglobulins and T cell receptorsis found in the CDRs, these regions are sometimes referred to ashypervariable regions. Among these, CDR3 shows the greatest variabilityas it is encoded by a recombination of VJ in the case of a light chainregion and VDJ in the case of heavy chain regions. The tertiarystructure of an antibody is important to analyze and design newantibodies.

As used herein, the terms “proteins” and “polypeptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein” and“polypeptide” refer to a polymer of protein amino acids, includingmodified amino acids (e.g., phosphorylated, glycated, glycosylated,etc.) and amino acid analogs, regardless of its size or function.“Protein” and “polypeptide” are often used in reference to relativelylarge polypeptides, whereas the term “peptide” is often used inreference to small polypeptides, but usage of these terms in the artoverlaps. The terms “protein” and “polypeptide” are used interchangeablyherein when referring to an encoded gene product and fragments thereof.Thus, exemplary polypeptides or proteins include gene products,naturally occurring proteins, homologs, orthologs, paralogs, fragmentsand other equivalents, variants, fragments, and analogs of theforegoing. The antibodies of the present invention are polypeptides, aswell the antigen-binding fragments and fragments thereof.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition that specifically binds to a single epitope ofthe antigen.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Other forms of “chimericantibodies” are those in which the class or subclass has been modifiedor changed from that of the original antibody. Such “chimeric”antibodies are also referred to as “class-switched antibodies.” Methodsfor producing chimeric antibodies involve conventional recombinant DNAand gene transfection techniques now well known in the art.

In a preferred embodiment, the present disclosure provides aCD30-antibody covalently linked to a CD3 antibody.

CD30 Antibodies or Antigen Binding Fragments Used in the BispecificAntibodies

The portion of the bispecific antibody that specifically and selectivelybinds to CD30 may be an antibody, e.g., monoclonal antibody or antigenbinding fragment thereof. The CD30 binding antibody or antigen bindingfragment thereof is capable of selectively binding to human CD30, andhaving a different binding specificity to CD30 than the known anti-CD30antibody AC10 (monoclonal antibody in brentuximab). By “selectively” or“specifically” we mean an antibody capable of binding human CD30 butdoes not bind to other CD molecules or other cell surface proteins. Bybinding, we mean that the antibodies are capable of detection at a giventissue's extracellular membrane by standard methods (e.g., tissuesection immunofluorescence assays or flow cytometry).

In one embodiment, the CD30 binding portion of the bispecific antibodyis a monoclonal antibody (MAbs) that target CD30 and derivativesthereof. Suitable monoclonal antibodies include, but are not limited to,monoclonal antibodies 8D10, 10C2, 12B1, 13H1, and 15B8 produced fromhybridoma cell lines as described herein. The monoclonal antibodies usedin the bispecific antibodies described herein are able to specificallyand selectively bind to CD30 (as demonstrated in FIG. 1 ). Each of fivehybridoma clones, designated as 8D10, 10C2, 12B1, 13H1, and 15B8, boundto CD30+ but not CD30− cell lines indicating specificity for theselected antigen CD30.

In one embodiment, the CD30 binding antibody or antigen binding fragmentthereof capable of binding human CD30 comprising, consisting orconsisting essentially of: (a) a light chain variable domain comprisinga CDRL1 region of SEQ ID NO:2 or a sequence with at least 85% similarityto SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3 or a sequence with atleast 85% similarity to SEQ ID NO:3, and a CDRL3 region of SEQ ID NO:4or a sequence with at least 85% similarity to SEQ ID NO:4 and a heavychain variable domain comprising a CDRH1 region of SEQ ID NO:6 or asequence with at least 85% similarity to SEQ ID NO:6, a CDRH2 region ofSEQ ID NO:7 or a sequence with at least 85% similarity to SEQ ID NO:7,and a CDRH3 region of SEQ ID NO:8 or a sequence with at least 85%similarity to SEQ ID NO:8; (b) a light chain variable domain comprisinga CDRL1 region of SEQ ID NO:10 or a sequence with at least 85%similarity to SEQ ID NO:10, a CDRL2 region of SEQ ID NO:11 or a sequencewith at least 85% similarity to SEQ ID NO: 11, and a CDRL3 region of SEQID NO:12 or a sequence with at least 85% similarity to SEQ ID NO: 12 anda heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:14or a sequence with at least 85% similarity to SEQ ID NO:14, a CDRH2region of SEQ ID NO:15 or a sequence with at least 85% similarity to SEQID NO:15 and a CDRH3 region of SEQ ID NO:16 or a sequence with at least85% similarity to SEQ ID NO:16, (c) a light chain variable domaincomprising a CDRL1 region of SEQ ID NO: 18 or a sequence with at least85% similarity to SEQ ID NO: 18, a CDRL2 region of SEQ ID NO:19 or asequence with at least 85% similarity to SEQ ID NO:19, and a CDRL3region of SEQ ID NO:20 or a sequence with at least 85% similarity to SEQID NO:20 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:22 or a sequence with at least 85% similarity to SEQ ID NO:22,a CDRH2 region of SEQ ID NO:23 or a sequence with at least 85%similarity to SEQ ID NO:23, and a CDRH3 region of SEQ ID NO:24 or asequence with at least 85% similarity to SEQ ID NO:24, (d) a light chainvariable domain comprising a CDRL1 region of SEQ ID NO:26 or a sequencewith at least 85% similarity to SEQ ID NO:26, a CDRL2 region of SEQ IDNO:27 or a sequence with at least 85% similarity to SEQ ID NO:27, and aCDRL3 region of SEQ ID NO:28 or a sequence with at least 85% similarityto SEQ ID NO:28 and a heavy chain variable domain comprising a CDRH1region of SEQ ID NO:30 or a sequence with at least 85% similarity to SEQID NO:30, a CDRH2 region of SEQ ID NO:31 or a sequence with at least 85%similarity to SEQ ID NO:31, and a CDRH3 region of SEQ ID NO:32 or asequence with at least 85% similarity to SEQ ID NO:32, or (e) a lightchain variable domain comprising a CDRL1 region of SEQ ID NO:34 or asequence with at least 85% similarity to SEQ ID NO:34 or a sequence withat least 85% similarity to SEQ ID NO:34, a CDRL2 region of SEQ ID NO:35or a sequence with at least 85% similarity to SEQ ID NO:35, and a CDRL3region of SEQ ID NO:36 or a sequence with at least 85% similarity to SEQID NO:36 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:38 or a sequence with at least 85% similarity to SEQ ID NO:38,a CDRH2 region of SEQ ID NO:39 or a sequence with at least 85%similarity to SEQ ID NO:39, and a CDRH3 region of SEQ ID NO:40 or asequence with at least 85% similarity to SEQ ID NO:40.

In another embodiment, the CD30 binding antibody or antigen bindingfragment thereof capable of binding human CD30 comprising, consisting orconsisting essentially of: a heavy and a light chain, wherein theantigen binding domain formed by the heavy and light chain is able tobind specifically to human CD30.

In one embodiment, the anti-CD30 antibody or antigen-binding fragmentthereof comprises, consists essentially of or consists of a light chaincomprising SEQ ID NO: 1 or a sequence with at least 85% similarity toSEQ ID NO: 1, and a heavy chain comprising SEQ ID NO:5 or a sequencewith at least 85% similarity to SEQ ID NO:5.

In another embodiment, the isolated anti-CD30 antibody orantigen-binding fragment thereof comprises, consists essentially of orconsists of a light chain comprising SEQ ID NO:9 or a sequence with atleast 85% similarity to SEQ ID NO:9, and a heavy chain comprising SEQ IDNO:13 or a sequence with at least 85% similarity to SEQ ID NO:13.

In another embodiment, the isolated anti-CD30 antibody orantigen-binding fragment thereof comprises, consists essentially of orconsists of a light chain comprising SEQ ID NO:17 or a sequence with atleast 85% similarity to SEQ ID NO:17, and a heavy chain comprising SEQID NO:21 or a sequence with at least 85% similarity to SEQ ID NO:21.

In another embodiment, the isolated anti-CD30 or antigen-bindingfragment thereof comprises, consists essentially of or consists of alight chain comprising SEQ ID NO:25 or a sequence with at least 85%similarity to SEQ ID NO:25, and a heavy chain comprising SEQ ID NO:29 ora sequence with at least 85% similarity to SEQ ID NO:29; and

In another embodiment, the isolated anti-CD30 antibody orantigen-binding fragment thereof comprises, consists essentially of orconsists of a light chain comprising SEQ ID NO:33 or a sequence with atleast 85% similarity to SEQ ID NO:33, and a heavy chain comprising SEQID NO:37 or a sequence with at least 85% similarity to SEQ ID NO:37.

In one embodiment, the CD30 binding antibody or antigen binding fragmentthereof capable of binding human CD30 comprising, consisting orconsisting essentially of: (a) a light chain variable domain comprisinga CDRL1 region of SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3, and aCDRL3 region of SEQ ID NO:4 and a heavy chain variable domain comprisinga CDRH1 region of SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7, and aCDRH3 region of SEQ ID NO:8; (b) a light chain variable domaincomprising a CDRL1 region of SEQ ID NO:10, a CDRL2 region of SEQ IDNO:11, and a CDRL3 region of SEQ ID NO: 12 and a heavy chain variabledomain comprising a CDRH1 region of SEQ ID NO: 14, a CDRH2 region of SEQID NO: 15 and a CDRH3 region of SEQ ID NO: 16, (c) a light chainvariable domain comprising a CDRL1 region of SEQ ID NO:18, a CDRL2region of SEQ ID NO: 19, and a CDRL3 region of SEQ ID NO:20 and a heavychain variable domain comprising a CDRH1 region of SEQ ID NO:22, a CDRH2region of SEQ ID NO:23, and a CDRH3 region of SEQ ID NO:24, (d) a lightchain variable domain comprising a CDRL1 region of SEQ ID NO:26, a CDRL2region of SEQ ID NO:27, and a CDRL3 region of SEQ ID NO:28 and a heavychain variable domain comprising a CDRH1 region of SEQ ID NO:30, a CDRH2region of SEQ ID NO:31, and a CDRH3 region of SEQ ID NO:32, or (e) alight chain variable domain comprising a CDRL1 region of SEQ ID NO:34, aCDRL2 region of SEQ ID NO:35, and a CDRL3 region of SEQ ID NO:36 and aheavy chain variable domain comprising a CDRH1 region of SEQ ID NO:38, aCDRH2 region of SEQ ID NO:39, and a CDRH3 region of SEQ ID NO:40.

In one embodiment, the anti-CD30 antibody is monoclonal antibody 8D10.In another embodiment, the anti-CD30 antibody is monoclonal antibody10C2. In another embodiment, the anti-CD30 antibody is monoclonalantibody 12B1. In another embodiment, the anti-CD30 antibody ismonoclonal antibody 13H1. In another embodiment, the anti-CD30 antibodyis monoclonal antibody 15B8. In a preferred embodiment, the anti-CD30antibody is monoclonal antibody 8D10 or 10C2.

In some embodiments, the anti-CD30 antibody or antigen-binding fragmentthereof is selected from the group consisting of a monoclonal antibody,a humanized antibody, a single chain variable fragment (scFv) antibody,a single domain antibody, an antigen-binding fragment and a chimericantibody.

In some embodiments, the antibodies comprise a light and a heavy chainthat have substantial identity to the polypeptide sequences found in SEQID NOs: 1 and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively,or substantial identity in the CDR regions within the heavy and lightchain of the antibody or antigen-binding fragment thereof as describedherein.

In some embodiments, the antibodies have at least 85% identity to thelight chain and heavy chain found in SEQ ID NOs:1 and 5, 9 and 13, 17and 21, 25 and 29, 33 and 37 respectively, alternatively at least 90%sequence identity to the light chain and heavy chain found in SEQ IDNOs:1 and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively,alternatively at least 95% sequence identity to the light chain andheavy chain found in SEQ ID NOs:1 and 5, 9 and 13, 17 and 21, 25 and 29,33 and 37 respectively, alternatively at least 97% sequence identity tothe light chain and heavy chain found in SEQ ID NOs: 1 and 5, 9 and 13,17 and 21, 25 and 29, 33 and 37 respectively, alternatively at least 98%sequence identity to the light chain and heavy chain found in SEQ IDNOs:1 and 5, 9 and 13, 17 and 21, 25 and 29, 33 and 37 respectively,alternatively at least 100% sequence identity to the light chain andheavy chain found in SEQ ID NOs:1 and 5, 9 and 13, 17 and 21, 25 and 29,33 and 37 respectively.

In some embodiments, the antibodies have at least 85% identity to theCDR domains described herein, alternatively at least 90% sequenceidentity, alternatively at least 95% sequence identity, alternatively atleast 97% sequence identity, alternatively at least 98% sequenceidentity, alternatively at least 100% sequence identity. In someembodiments, the antibody or antigen binding fragment thereof has atleast 85-100% sequence identity within CDRH1, CDRH2 and CDRH3 within SEQID NO:5 (e.g., SEQ ID Nos. 6-8), SEQ ID NO:13 (e.g., SEQ ID Nos: 14-16),SEQ ID NO:21 (e.g., SEQ ID Nos: 22-24), SEQ ID NO:29 (e.g., SEQ ID Nos:30-32), or SEQ ID NO:37 (e.g., SEQ ID NOS: 38-40) and/or at least85%-100% sequence identity within CDRL1, CDRL2 and CDRL3 within SEQ IDNO:1 (e.g. SEQ ID Nos. 2-4), SEQ ID NO:9 (e.g., SEQ ID Nos: 10-12), SEQID NO:17 (e.g., SEQ ID NOs. 18-20), SEQ ID NO:25 (e.g., SEQ ID Nos:26-28), or SEQ ID NO:33 (e.g., SEQ ID Nos: 34-36).

In one embodiment, the antibody or antigen binding fragment thereof hasat least 95-100% sequence identity within CDRH1, CDRH2 and CDRH3 withinSEQ ID NO:5 (e.g., SEQ ID Nos. 6-8), SEQ ID NO:13 (e.g., SEQ ID Nos:14-16), SEQ ID NO:21 (e.g., SEQ ID Nos: 22-24), SEQ ID NO:29 (e.g., SEQID Nos: 30-32), or SEQ ID NO:37 (e.g., SEQ ID NOS: 38-40) and/or atleast 95%-100% sequence identity within CDRL1, CDRL2 and CDRL3 withinSEQ ID NO:1 (e.g. SEQ ID Nos. 2-4), SEQ ID NO:9 (e.g., SEQ ID Nos:10-12), SEQ ID NO:17 (e.g., SEQ ID NOs. 18-20), SEQ ID NO:25 (e.g., SEQID Nos: 26-28), or SEQ ID NO:33 (e.g., SEQ ID Nos: 34-36).

In one embodiment, the antibody or antigen binding fragment thereof has100% sequence identity within CDRH1, CDRH2 and CDRH3 within SEQ ID NO:5(e.g., SEQ ID Nos. 6-8), SEQ ID NO:13 (e.g., SEQ ID Nos: 14-16), SEQ IDNO:21 (e.g., SEQ ID Nos: 22-24), SEQ ID NO:29 (e.g., SEQ ID Nos: 30-32),or SEQ ID NO:37 (e.g., SEQ ID NOS: 38-40) and/or 100% sequence identitywithin CDRL1, CDRL2 and CDRL3 within SEQ ID NO: 1 (e.g. SEQ ID Nos.2-4), SEQ ID NO:9 (e.g., SEQ ID Nos: 10-12), SEQ ID NO:17 (e.g., SEQ IDNOs. 18-20), SEQ ID NO:25 (e.g., SEQ ID Nos: 26-28), or SEQ ID NO:33(e.g., SEQ ID Nos: 34-36).

T Cell or NK Surface Antigen Antibodies or Antigen Binding FragmentsUsed in the Bispecific Antibodies

The portion of the bispecific antibody that specifically and selectivelybinds to T cell or NK cell surface may be an antibody, e.g., monoclonalantibody or antigen binding fragment thereof. T cell surface markers areknown in the art and include, but are not limited to, for example, CD3.In one embodiment, the suitable anti-T cell surface antibodies are knownin the art and include, but are not limited to, e.g., anti-CD3 antibodyor antigen binding fragment thereof. Suitably, the anti-T cell surfaceantibody is an anti-CD3 antibody.

The anti-CD3 antibody to be used in the present invention may be anyantibody as long as it is specific for CD3. The CD3 antibody may be aCD3 monoclonal antibody, for example OKT3. OKT3, a murine IgG2a mAbdirected against the ε-chain of the CD3 complex on human T lymphocytes(Salmeron et al., J. Immunol. 147 (1991), 3047-3052) and produced by ahybridoma with the ATCC deposit number of CRL 8001.

The NK cell surface antigen may by an NK surface antigen known in theart, for example, CD16. In one embedment, a bispecific antibodycomprising an anti-CD30 antibody described herein and an anti-CD16antibody or antigen binding fragment thereof is contemplated.

The polypeptide and nucleic acids described herein encompass those towhich conservative modifications have been made. The terms “conservativemodification” or “conservative sequence modification” refer to an aminoacid modification that does not significantly alter the bindingcharacteristics of an antibody or antibody fragment containing an aminoacid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto the antibodies or antibody fragments of the present invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutions arethose in which the amino acid residue is replaced by an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the related art. Thesefamilies include, but are not limited to, basic side chains (e.g.,lysine (Lys, L), arginine (Arg), histidine (His, H); acidic side chains(e.g., aspartic acid (Asp, D), glutamic acid (Glu, E)), uncharged polarside chains (e.g., asparagine (Asn, N), glutamine (Gln, Q), serine (Ser,S), threonine (Thr, T), tyrosine (Tyr, Y), nonpolar side chains (e.g.,alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile,I), proline (Pro, P), phenylalanine (Phe, F), methionine (Met, M),Glycine (Gly, G), Cysteine (Cys, C)), beta-branched side chains (e.g.,leucine (L), valine (V), isoleucine (I)). Thus, one or more amino acidresidues within the antibody or antigen binding fragment thereof of thepresent invention may be replaced by other amino acid residues from thesame side chain family, and the altered antibodies or antibody fragmentsthereof may be tested using the functional assays described herein.Suitably, conservative changes may even be made in the CDR region andnot alter the functional binding of the antibody or antigen bindingfragment thereof, which can be tested by the methods described herein.

Protein and nucleic acid sequence identities are evaluated using theBasic Local Alignment Search Tool (“BLAST”) which is well known in theart (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). TheBLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis known or obtained from a protein or nucleic acid sequence database.Preferably, the statistical significance of a high-scoring segment pairis evaluated using the statistical significance formula (Karlin andAltschul, 1990), the disclosure of which is incorporated by reference inits entirety. The BLAST programs can be used with the default parametersor with modified parameters provided by the user.

“Percentage of sequence identity” or “sequence similarity” is determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 85% sequenceidentity. Alternatively, percent identity can be any integer from 75% to100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%compared to a reference sequence using the programs described herein;preferably BLAST using standard parameters, as described. These valuescan be appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like.

“Substantial identity” of amino acid sequences for purposes of thisinvention normally means polypeptide sequence identity of at least 85%.Preferred percent identity of polypeptides can be any integer from 85%to 100%. More preferred embodiments include at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

One may wish to express the bispecific antibody as a fusion proteinlinking the anti-CD30 antibody to the anti-T cell surface antigenantibody (e.g., anti-CD3 antibody). For example, one may wish to expressthe bispecific antibody of the present invention with a protein linkerbetween the two antibodies. Standard molecular biology techniques (e.g.,restriction enzyme based sub-cloning, or homology based sub-cloning)could be used to place the DNA sequence encoding the first anti-CD30antibody in frame with the second anti-CD3 antibody to form a singlebispecific antibody. The fusion protein is then produced as one peptidein a host cell (e.g., yeast, bacteria, insect, or mammalian cell) andpurified before use. Note the therapeutic does not need to be a wholeprotein. For example, it can be a single peptide chain comprising the VHand VL sequences of the CD30 antibody linked to the VH and VL sequencesof the CD3 antibody.

Making of Bispecific Antibodies

Conventional linking methods of linking polypeptides, in particular anantibody, are known in the art (e.g., See TERNYNCK and AVRAMEAS, 1987,“Techniques immunoenzymatiques” Ed. INSERM, Paris or G. T. Hermanson,Bioconjugate Techniques, 2010, Academic Press). Many chemicalcross-linking methods are also known in the art. Cross-linking reagentsmay be homobifunctional (i.e., having two functional groups that undergothe same reaction) or heterobifunctional (i.e., having two differentfunctional groups). Numerous cross-linking reagents are commerciallyavailable. Detailed instructions for their use are readily availablefrom the commercial suppliers. A general reference on polypeptidecross-linking and conjugate preparation is: WONG, Chemistry of proteinconjugation and cross-linking, CRC Press (1991).

The anti-CD30 and anti-T cell/NK cell antibody (e.g., anti-CD3 antibody)may be covalently linked as depicted in FIGS. 14A-14F and as describedin “CD20-Targeted T Cells after Stem Cell Transplantation for High Riskand Refractory Non-Hodgkin's Lymphoma” Lum, Lawrence G. et al., Biologyof Blood and Marrow Transplantation, Volume 19, Issue 6, 925-933,incorporated by reference in its entirety. Briefly, the process ofheteroconjugation of anti-CD3 with anti-CD30 antibody is shown in FIGS.14A-14F. FIG. 14A shows one embodiment of how the antibodies may beconjugated together, by the end of the Fc portions. FIG. 14B shows amodel method of covalently linking the antibodies. Step 1 showscross-linking of Traut's reagent to anti-CD3 (OKT3) mAb and thecross-linking of Sulpho-SMCC to anti-CD30; step 2 shows theheteroconjugation of the cross-linked anti-CD3 with the cross-linkedanti-CD30; and step 3 shows formation of anti-CD3-anti-CD20 BiAb(CD30Bi).

Other suitable methods of covalently linking two antibodies or antigenbinding fragments thereof are known in the art. For example, thebispecific antibodies of the invention can be produced by a process inwhich two immunoglobulin molecules are linked together using a crosslinking agent such as N-succinimidyl 3-(2-pyridyldithiol)-propionate,S-acetylmercaptosuccinic acid anhydride or the like (J. Exp. Med., 163,166 (1986)) or by a process in which Fab fragments of immunoglobulinmolecules are linked together (Eur. J. Immunol., 19, 1437 (1989)).

Methods of preparing bispecific antibodies of the present inventioninclude those described in WO 2008119353 (Genmab), WO 2011131746(Genmab) and reported by van der Neut-Kolfschoten et al. (Science. 2007Sep. 14; 317(5844): 1554-7). Examples of other platforms useful forpreparing bispecific antibodies include but are not limited to BiTE(Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineeredIgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

In addition, methods such as the hybrid hybridoma and chemicalconjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26: 649)can also be used. Co-expression in a host cell of two antibodies,consisting of different heavy and light chains, leads to a mixture ofpossible antibody products in addition to the desired bispecificantibody, which can then be isolated by, e.g., affinity chromatographyor similar methods.

Strategies favoring the formation of a functional bispecific, product,upon co-expression of different antibody constructs can also be used,e.g., the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hydridomas producing different antibodiesleads to a limited number of heterodimeric proteins because ofpreferential species-restricted heavy/light chain pairing. Anotherstrategy to promote formation of heterodimers over homodimers is a“knob-into-hole” strategy in which a protuberance is introduced on afirst heavy-chain polypeptide and a corresponding cavity in a secondheavy-chain polypeptide, such that the protuberance can be positioned inthe cavity at the interface of these two heavy chains so as to promoteheterodimer formation and hinder homodimer formation. “Protuberances”are constructed by replacing small amino-acid side-chains from theinterface of the first polypeptide with larger side chains. Compensatory“cavities” of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino-acidside-chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459(Chugai) and WO 2009089004 (Amgen) describe other strategies forfavoring heterodimer formation upon co-expression of different antibodydomains in a host cell. In these methods, one or more residues that makeup the CH3-CH3 interface in both CH3 domains are replaced with a chargedamino acid such that homodimer formation is electrostaticallyunfavorable and heterodimerization is electrostatically favorable.WO2007110205 (Merck) describe yet another strategy, wherein differencesbetween IgA and IgG CH3 domains are exploited to promoteheterodimerization.

Another in vitro method for producing bispecific antibodies has beendescribed in WO 2008119353 (Genmab), wherein a bispecific antibody isformed by “Fab-arm” or “half-molecule” exchange (swapping of a heavychain and attached light chain) between two monospecific IgG4- orIgG4-like antibodies upon incubation under reducing conditions. Theresulting product is a bispecific antibody having two Fab arms which maycomprise different sequences.

Further embodiments provide an isolated nucleic acid that encodes forthe bispecific antibodies or antigen binding fragment thereof describedabove. Some embodiments provide an isolated polynucleotide encoding abispecific antibody described herein. As used herein, term “nucleicacid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) orribonucleic acids (RNA) and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), alleles, orthologs, SNPs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol.Cell. Probes 8:91-98 (1994)).

A recombinant expression cassette comprising a polynucleotide encodingthe bispecific antibody or antigen binding fragment thereof of thepresent invention is also contemplated. The polynucleotide may be underthe control of a transcriptional promoter allowing the regulation of thetranscription of said polynucleotide in a host cell. Said polynucleotidecan also be linked to appropriate control sequences allowing theregulation of its translation in a host cell.

The present invention also provides a expression vector or recombinantvector (e.g., a recombinant expression vector) comprising apolynucleotide encoding the bispecific antibodies according to thepresent invention. Advantageously, said recombinant expression vector isa recombinant expression vector comprising an “expression cassette” or“expression construct” according to the present invention. With theconstruct, the polynucleotides may operatively linked to atranscriptional promoter (e.g., a heterologous promoter) allowing theconstruct to direct the transcription of said polynucleotide in a hostcell. Such vectors are referred to herein as “recombinant constructs,”“expression constructs,” “recombinant expression vectors” (or simply,“expression vectors” or “vectors”).

The term vector includes a nucleic acid molecule capable of propagatinganother nucleic acid to which it is linked. The term includes the vectoras a self-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. Certain vectors are capable of directing the expression ofnucleic acids to which they are operatively linked. One type of vectoris a “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated, specifically exogenous DNAsegments encoding the antibodies or fragments thereof. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced. Other vectorscan be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome(e.g. lentiviral vectors). Vector includes expression vectors, such asviral vectors (e.g., replication defective retroviruses (includinglentiviruses), adenoviruses and adeno-associated viruses (rAAV)), whichserve equivalent functions. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated, specifically exogenous DNA segments encodingthe antibodies or fragments thereof. Another type of vector is a viralvector, wherein additional DNA segments may be ligated into the viralgenome. Certain vectors are capable of autonomous replication in a hostcell into which they are introduced. Other vectors can be integratedinto the genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome (e.g. lentiviralvectors). Vector includes expression vectors, such as viral vectors(e.g., replication defective retroviruses (including lentiviruses),adenoviruses and adeno-associated viruses (rAAV)), which serveequivalent functions.

The present invention also provides a host cell able to express thebispecific antibody described herein. In one embodiment, the host cellis a fused hybridoma cell of two hydridoma cells. In another embodiment,the host cell contains isolated nucleic acids or a recombinantexpression cassette or a recombinant expression vector according to thepresent invention able to express the bispecific antibody. The host cellcan be a prokaryotic or eukaryotic host cell. The host cell is capableof expressing the bispecific antibodies of the present invention.Suitable host cells include, but are not limited to, mammalian cells,bacterial cells and yeast cells. In some embodiments, the host cell maybe a eukaryotic cell. The terms “host cell” also includes a cell intowhich exogenous nucleic acid has been introduced, including the progenyof such cells. Host cells include “transformants” and “transformedcells”, which include the primar transformed cell and progeny derivedtherefrom without regard to the number of passages. Progeny may not becompletely identical in nucleic acid content to a parent cell, but maycontain mutations. Mutant progeny that have the same function orbiological activity as screened or selected for in the originallytransformed cell are included herein.

In other embodiments, the invention includes a purified and isolatedhost cell comprising an expression vector containing an isolated nucleicacid capable of encoding the bispecific antibody. It should beappreciated that the host cell can be any cell capable of expressingantibodies, for example fungi; mammalian cells; insect cells, using, forexample, a baculovirus expression system; plant cells, such as, forexample, corn, rice, Arabidopsis, and the like. See, generally, Verma,R. et al., J Immunol Methods. 1998 Jul. 1; 216(1-2):165-81.

The bispecific can be wholly or partially synthetically produced thatare used to make the bispecific antibody. Thus the antibody may be fromany appropriate source, for example recombinant sources and/or producedin transgenic animals or transgenic plants. Thus, the bispecificantibody molecules can be produced in vitro or in vivo. Preferably theanti-CD30 or anti-CD3 antibody or antibody fragment comprises at leastthe heavy chain variable region (VH) which generally comprises theantigen binding site. In preferred embodiments, the antibody or antibodyfragment comprises the heavy chain variable region and light chainvariable region (VL). The bispecific antibody comprising the antibody orantibody fragment can be made that comprises all or a portion of a heavychain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2,IgE, IgM or IgD constant region.

Furthermore, the antibody or antibody fragment can comprise all or aportion of a kappa light chain constant region or a lambda light chainconstant region. All or part of such constant regions may be producedwholly or partially synthetic. Appropriate sequences for such constantregions are well known and documented in the art.

In some embodiments, the disclosure provides a pharmaceuticalcomposition comprising the isolated bispecific antibody or antigenbinding fragment thereof specific for human CD30 and a T cell surfaceantigen (e.g., CD3). In a preferred embodiment, the composition furtherincludes a suitable carrier, preferably a pharmaceutically acceptablecarrier. Compositions are provided that include one or more of thedisclosed bispecific antibodies. The compositions can be prepared inunit dosage forms for administration to a subject. The amount and timingof administration are at the discretion of the treating clinician toachieve the desired outcome. The bispecific antibody can be formulatedfor systemic or local (such as intravenous, intrathecal) administration.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the antibody together with apharmaceutically-acceptable carrier. “Pharmaceutically acceptable”carriers are known in the art and include, but are not limited to, forexample, suitable diluents, preservatives, solubilizers, emulsifiers,liposomes, nanoparticles and adjuvants. Pharmaceutically acceptablecarriers are well known to those skilled in the art and include, but arenot limited to, 0.01 to 0.1 M and preferably 0.05M phosphate buffer or0.9% saline. Additionally, such pharmaceutically acceptable carriers maybe aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include isotonic solutions,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media.

Pharmaceutical compositions of the present disclosure may includeliquids or lyophilized or otherwise dried formulations and may includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e. g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), covalentattachment of polymers such as polyethylene glycol to the protein,complexation with metal ions, or incorporation of the material into oronto particulate preparations of polymeric compounds such as polylacticacid, polyglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, milamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance. Controlled or sustained release compositionsinclude formulation in lipophilic depots (e.g., fatty acids, waxes,oils).

In some embodiments, the compositions comprise a pharmaceuticallyacceptable carrier, for example, buffered saline, and the like. Thecompositions can be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableadditional substances as required to approximate physiologicalconditions such as a pH adjusting and buffering agent, toxicityadjusting agents, such as, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate, and the like.

In some embodiments, the antibodies are provided in lyophilized form andrehydrated with sterile water or saline solution before administration.In some embodiments, the antibodies are provided in sterile solution ofknown concentration. In some embodiments, the antibody composition maybe added to an infusion bag containing 0.9% sodium chloride, USP and insome cases, administered in a dosage of from 0.5 to 15 mg/kg of bodyweight.

Methods of Use

The bispecific antibodies described herein are able to stimulate a Tcell or NK cell response to a specific CD30+ tumor cell. The bispecificantibody bring T-cell via its CD3 receptor into close contact with thetumor cell (via its CD30 antigen) to form an immunological synapse,which generates a strong activating signal cell mediated signal. Thebispecific antibody allows the T-cells to be activated at the site oftumor cells allowing for a T-cell mediated response against the CD30+tumor cell.

In one embodiment, this activating signal induces the transport ofcytotoxic granules to the cell surface where they release perforin andgranzyme in the vicinity of the tumor cell. For example, granulescontaining cell lysing components, such as perforin, granzyme andlysosomal enzymes, are transported towards the cell membrane of theT-cell. Once at the surface and subsequently secreted into theextracellular matrix. Perforin causes the formation of pores in thetarget cell, thereby facilitating the entry of the cell lysingcomponents. This set of activities leads to tumor cell apoptosis.

In another embodiment, the present disclosure provides a method ofinhibiting or reducing growth of a tumor cell expressing CD30,comprising contacting the tumor cell with an effective amount of thebispecific antibody of such that the growth of the cell is inhibited. Insome embodiments, inhibiting cell growth includes killing orapoptosising the cancer cell. In some embodiments, the method includesreducing, inhibiting or preventing growth of tumor cells.

As used herein, the term “inhibits proliferation” (e.g. referring tocells, such as tumor cells) is intended to include any substantialdecrease in the cell proliferation when contacted with a bispecificantibody as compared to the proliferation of the same cells not incontact with the bispecific antibody, e.g., the inhibition ofproliferation of a cell culture by at least about 10%, at least about20% or at least about 30%.

In some embodiments, the antibodies of the invention specifically bindCD30 and T cells (via CD3) and exert cytostatic and cytotoxic effects onmalignant cells in cancer (e.g., Hodgkin's lymphoma).

One embodiment of the present invention provides a method of treating apatient having a CD30⁺ cancer, the method comprising administering atherapeutically effective amount of the isolated bispecific antibodydescribed herein capable of simultaneously binding human CD30 and T cellsurface antigen as described herein to treat the cancer.

For purposes of the present invention, “treating” or “treatment”describes the management and care of a subject for the purpose ofcombating the disease, condition, or disorder. Treating includes theadministration of an antibody of present invention to prevent the onsetof the symptoms or complications, alleviating the symptoms orcomplications, or eliminating the disease, condition, or disorder.

The term “treating” can be characterized by one or more of thefollowing: (a) the reducing, slowing or inhibiting the growth of cancer,including reducing slowing or inhibiting the growth of cancer cells; (b)preventing the further growth of tumors; (c) reducing or preventing themetastasis of cancer within a patient, and (d) reducing or amelioratingat least one symptom of the cancer. In some embodiments, the optimumeffective amounts can be readily determined by one of ordinary skill inthe art using routine experimentation.

In another embodiment, the treatment can result in cell-cycle inhibitionof tumor cells (i.e., cytostasis). In another embodiment, the treatmentcan result in a T-cell mediated killing of the tumor cells.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” refer to the quantity of active therapeutic agent oragents sufficient to yield a desired therapeutic response without undueadverse side effects such as toxicity, irritation, or allergic response.The specific “effective amount” will, obviously, vary with such factorsas the particular condition being treated, the physical condition of thesubject, the type of animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compounds or itsderivatives.

In some embodiments, the bispecific antibody of the present invention isused for treatment in addition to standard treatment options, forexample surgery and radiation therapy. In some embodiments, theantibodies of the present disclosure are used in combination therapy,e.g. therapy including one or more different anti-cancer agents.

Suitable CD30⁺ cancers include, hematologic malignancies, for example,Hodgkins lymphoma, anaplastic large cell lymphoma, acute myeloidleukemia (AML), ovarian cancer, mesothelioma, skin squamous cellcarcinoma, triple negative breast cancer, pancreatic cancer, small celllung cancer, anal cancer, and thyroid carcinoma, among others. CD30 hasalso been shown to be expressed on a subset of non-Hodgkin's lymphomas(NHL), including Burkitt's lymphoma, anaplastic large-cell lymphomas(ALCL), cutaneous T-cell lymphomas, nodular small cleaved-celllymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert'slymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL),adult T-cell leukemia (T-ALL), and entroblastic/centrocytic (cb/cc)follicular lymphomas, along with embryonal carcinomas, nonembryonalcarcinomas, malignant melanomas, and mesenchymal tumors. As such, thepresent methods may be used to treat any cancer in which CD30⁺ tumorcells are found.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, intraaural administration,rectal administration, sublingual administration, buccal administration,and parenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, intradermal administration, intrathecal administrationand subcutaneous administration. Administration can be continuous orintermittent. In various aspects, a preparation can be administeredtherapeutically; that is, administered to treat an existing disease orcondition. In a preferred embodiment, the administration is intravenousadministration.

The present invention also includes anti-CD3/anti-CD30 bispecificantibodies which exhibit one or more characteristics selected from thegroup consisting of: (a) inducing T cell proliferation; (b) activatingT-cells, inducing IFN-gamma release; (c) inducing T-cell mediatedcytotoxicity on anti-CD30+ tumor cells; (d) increasing CD30+ tumor celldeath; and (e) decreasing CD30+ tumor volume.

Kits

In some embodiments, kits for carrying out the methods described hereinare provided. The kits provided may contain the necessary componentswith which to carry out one or more of the above-noted methods. In oneembodiment, kits for treating a subject with a CD30 cancer are provided.The kit comprises a bispecific antibody described herein andinstructions for use. Further, the kit may comprise a pharmaceuticallyacceptable carrier and instructions for use.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

It should be apparent to those skilled in the art that many additionalmodifications beside those already described are possible withoutdeparting from the inventive concepts. In interpreting this disclosure,all terms should be interpreted in the broadest possible mannerconsistent with the context. Variations of the term “comprising” shouldbe interpreted as referring to elements, components, or steps in anon-exclusive manner, so the referenced elements, components, or stepsmay be combined with other elements, components, or steps that are notexpressly referenced. Embodiments referenced as “comprising” certainelements are also contemplated as “consisting essentially of” and“consisting of” those elements. The term “consisting essentially of” and“consisting of” should be interpreted in line with the MPEP and relevantFederal Circuit interpretation. The transitional phrase “consistingessentially of” limits the scope of a claim to the specified materialsor steps “and those that do not materially affect the basic and novelcharacteristic(s)” of the claimed invention. “Consisting of” is a closedterm that excludes any element, step or ingredient not specified in theclaim. For example, with regard to sequences “consisting of” refers tothe sequence listed in the SEQ ID NO. and does refer to larger sequencesthat may contain the SEQ ID as a portion thereof.

The invention will be more fully understood upon consideration of thefollowing non-limiting examples.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

The invention will be more fully understood upon consideration of thefollowing non-limiting examples.

EXAMPLES Example 1: Anti-CD30 Monoclonal Antibody Production

We have employed hybridoma technology to generate novel murineanti-human CD30 mAbs. Mice were immunized and boosted with purifiedhuman GST-tagged CD30. Mouse spleen cells were then harvested and fusedwith myeloma cells to generate antibody secreting hybridoma. Hybridomasupernatants were screened for specificity to purified CD30 protein byELISA, and GST specific clones were eliminated. Fifteen anti-human CD30hybridoma cell lines were made, five were selected for further analysis.Specific binding of our mAbs to cell-surface expressed CD30 was assessedby flow cytometry (FIG. 1A-C) using 293T cells (CD30−), lentiviraltransduced 293T cells expressing huCD30, and K562 cells which arenaturally CD30+. The 293T (CD30−), 293T LV huCD30 (transduced with humanCD30), SU-DHL-1 (lymphoma), RPMI.6666 (lymphoma) or K562 (CML) celllines were incubated with each antibody in the form of unpurifiedhybridoma supernatant, and then incubated with an Alexa Fluor647-labeled anti-mouse IgG antibody. Cell-associated fluorescence wasdetermined by FACS. FIG. 2 demonstrates binding of purified antibodiesto cells with CD30 surface expression.

Each of five hybridoma clones, designated as 8D10, 10C2, 12B1, 13H1, and15B8, bound to CD30+ but not CD30− cell lines indicating specificity forthe selected antigen. See FIG. 1 and FIG. 2 .

All candidates show specific binding to CD30 and have been DNA andprotein sequenced. The percentage identity between the heavy and lightchains of the monoclonal antibodies selected were compared with theresults shown in FIG. 3A and FIG. 3B.

CD30 mAb Binding—ELISA

Microtiter plates were coated with recombinant CD30-GST fusion protein.Wells were blocked with 5% bovine serum albumin (BSA) solution. Purified8D10, 10C2, 12B1, 13H1, 15B8, BY88 (commercial anti-CD30 antibody), AC10(commercial anti-CD30 antibody) or MI15 (commercial anti-CD138 antibody)were added and incubated at varying concentrations. Wells were detectedby incubating with an alkaline phosphatase-labeled anti-mouse IgGantibody. The plate was developed with pNPP (p-nitrophenyl phosphate).The optical density at 405 was determined using a plate reader and theresults are shown in FIG. 4 .

CD30 Mab Epitope Studies.

CD30+SU-DHL-1 cells were blocked with unlabeled 8D10, 10C2, 12B1, 13H1,15B8, or AC10 at 6 serial dilutions. The blocked cells were thenincubated with fluorescently-labeled 8D10, 10C2, 12B1, 13H1, 15B8, orAC10. Excess labelled antibody was washed from the cells, and thecell-associated fluorescence was determined by FACS. Data is shown as acurve, plotted against increasing concentrations of blocking ab (FIGS.5-10 ), and as a bar graph showing that values at the maximumconcentration (2.5 ug) of blocking antibody (FIGS. 11-13 ).

All five antibodies have unique light and heavy chain sequences. Thesesequences also differ from the FDA-approved anti-CD30 antibody AC10(Brentuximab vedotin). All five antibodies bind to CD30, as indicated byFACS and ELISA assays. All five antibodies bind to an epitope that isdifferent from AC10 (Brentuximab). 8D10, 12B1, 13H1, 15B8 bind to thesame or similar epitope as each other while 10C2 binds to an epitopethat is distinct both from AC10, and from the other four novelantibodies reported here. FIGS. 19A-19B demonstrates the bindingspecificity of the new CD30 antibodies.

All antibodies bind an epitope distinct from Brentuximab vedotin.

Example 2: Bispecific CD30-CD3 Antibody as Immunotherapy for HodgkinLymphoma

This Example demonstrates the ability to make bispecific antibodies thatbind to CD30 and to T cells (via CD3) to produce an anti-cancer effectfor Hodgkin's Lymphoma. Two anti-CD30 clones (8D10 and 10C2) werecovalently heteroconjugated to an anti-T cell antibody (CD3) (OKT3).

FACS was used to demonstrates that the hetero-conjugated CD30 antibodiesbind to both a CD30+ Hodgkin's Lymphoma cell line (SU-DHL-1 cells, FIG.14B) and to primary human T cells (FIG. 14B) for both the 8D10biAb (red)and 10C2 biAb (blue).

The ability of the biAb to bind T cells was also confirmed usingfluorescently labelled T cells and labelled CD30+ tumor cells bymeasuring the formation of T cell-tumor cell dual-color conjugates,which only in the presence of biAb (FIG. 14E top right corner). Theability of the biAb to conjugate T cells and tumor cells was quantitatedas shown in FIG. 17D.

The ability to stimulate the effector function of T cells was measuredby production of pro-inflammatory cytokines IFN-γ and IL-2. As shown inFIGS. 16A-18B, 8D10 biAb or 10C2 biAb-armed T cells produce thepro-inflammatory cytokines IFN-γ (A) and IL-2 (B) when co-cultured withCD30+ tumor cells, indicating that binding of T cells to the biAb issufficient to trigger T cell activation and cytotoxicity. Armed T cellscocultured with CD30− cells have much lower cytokine productiondemonstrating the specificity of this approach. The cytotoxicity of thebiAbs was also confirmed by cell death in the presence of the 8D10 and10C2 biAb-armed T cells against CD30+ tumor cells. CD30+ tumor celllines SU-DHL-1, RPMI6666 and Raji LV30 (Raji cells transduced with CD30lentivirus) are killed with varying efficacy by 8D10 and 10C2 biAb-armedT cells as demonstrated in FIGS. 17A-17D. Non-transduced Raji cells(FIG. 18A, CD30−) are not killed by the armed T cells. FIG. 18Bdemonstrates that the CD30 expression on activated T cells is low enoughto avoid elimination by the 8D10 and 10C2 biAb armed T cells.

As such, this Example demonstrates the ability and specificity of thebiAb to activate T cells and kill tumor cells. The bispecific antibodyresults in the release of proinflammatory cytokines, which triggers Tcell cytotoxicity specifically against CD30+ cells, while sparring CD30−cells. Also, T cell fratricide as a result of low CD30 expression onactivated T cells is not observed.

Each publication, patent, and patent publication cited in thisdisclosure is incorporated in reference herein in its entirety. Thepresent invention is not intended to be limited to the foregoingexamples, but encompasses all such modifications and variations as comewithin the scope of the appended claims.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“650053.00647_ST25.txt” which is 16.5 kb in size was created on Sep. 24,2019 and electronically submitted via EFS-Web herewith the applicationis incorporated herein by reference in its entirety.

Anti-CD30 mAb CDR 8D10-Light chain (SEQ ID NO: 1)DIVMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQFLIYAATSLADGVPSRF SGSGSGTKFSFKISSLQAEDFVSYYC QQLYSTPFT FGGGTKLEIK (CDRL1-SEQ ID NO: 2-underline; CDRL2-SEQID NO: 3-bold; CDRL3-SEQ ID NO: 4-bold/underline) 8D10-Heavy chain(SEQ ID NO: 5) QVQLQESGTELVKPGASVKLSCKASGYTFTSYWMHWMKQRPGQGLEWIGNINPSNGGTNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR RD YYYGSSYGFDVWGTGTTVTVSS (CDH1-SEQ ID NO: 6- underline; CDH2-SEQ ID NO: 7-bold; CDH3-SEQ ID NO: 8-bold/underline) 10C2-Light chain (SEQ ID NO: 9)DIVLTQTPLTLSVTIGQPASISCKSNQSLLDSYGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFP RTFGGGTKLEIK (CDRL1-SEQ ID NO: 10-underline;CDRL2-SEQ ID NO: 11-bold; CDRL3-SEQ ID NO: 12- bold/underline)10C2-Heavy chain (SEQ ID NO: 13)QVQLEQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGGTSYNQKFKGKATLTVDKSSSTACMELNCLTSEDSAVYYCTL GA YVVGQGTSVTVSS (CDH1-SEQ ID NO: 14-underline;CDH2-SEQ ID NO: 15-bold; CDH3-SEQ ID NO: 16- bold/underline)12B1-Light chain (SEQ ID NO: 17)DIVMTQTTASLSTSVGETVTITCRASGNLHSYLTWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKIDSLQPEDFGSYYC QHFWTTPFT FGSGTKLEIK (CDRL1-SEQ ID NO: 18-underline; CDRL2-SEQ ID NO: 19-bold; CDRL3-SEQ ID NO: 20-bold/ underline)12B-1Heavy chain (SEQ ID NO: 21)EVKLEESGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPTNGGTNYNEKFKSKATLTVDKSSRTAYMQLSSLTSGDSAVYYCAR RD FITTSGFAYWGQGTLVTVSA (CDH1-SEQ ID NO: 22-underline; CDH2-SEQ ID NO: 23-bold; CDH3-SEQ ID NO: 24-bold/underline)13H1 Light chain (SEQ ID NO: 25)DIVMTQTPKSMSMSVGERVTLSCKASENVGTYVSWYQQKPEQSPKVLIYGASNRFTGVPDRFTGSGSATDFTLTISSVQTEDLADYHC GQSYSYPLT FGAGTKLELK (CDRL1-SEQ ID NO: 26-underline; CDRL2-SEQID NO: 27-bold; CDRL3-SEQ ID NO: 28-bold/ underline) 13H1-Heavy chain(SEQ ID NO: 29) QVQLQQSGTELVKPGASVKLSCKASGHTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR RG YYGSSSYWSFDVWGTGTTVTVSS (CDH1-SEQ ID NO: 30-underline; CDH2-SEQ ID NO: 31-bold; CDH3-SEQ ID  NO: 32-bold/underline)15B8 Light chain (SEQ ID NO: 33)DIVMTQTPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRF SGSGSGTQYSLKINSLQPEDFGSYYC QHFWSTPFT FGSGTKLEIK (CDRL1-SEQ ID NO: 34-underline; CDRL2-SEQ ID NO: 35-bold; CDRL3-SEQ ID NO: 36-bold/ underline)15B8-Heavy chain (SEQ ID NO: 37)QVQLEQSGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAIYYCAR RN NYYASSPFAYWGQGTLVSVSA (CDH1-SEQ ID NO: 38-underline; CDH2-SEQ ID NO: 39-bold; CDH3-SEQ ID NO: 40-bold/underline

The invention claimed is:
 1. An isolated bispecific antibody capable ofbinding human CD30 and to CD3 comprising an anti-CD30 antibody orantigen binding portion thereof and an anti-CD3 antibody or antigenbinding portion thereof, wherein the anti-CD30 antibody or antigenbinding portion thereof comprises: (a) a light chain variable domaincomprising a CDRL1 region of SEQ ID NO:2 or a sequence with at least 85%similarity to SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3 or a sequencewith at least 85% similarity to SEQ ID NO:3, and a CDRL3 region of SEQID NO:4 or a sequence with at least 85% similarity to SEQ ID NO:4 and aheavy chain variable domain comprising a CDRH1 region of SEQ ID NO:6 ora sequence with at least 85% similarity to SEQ ID NO:6, a CDRH2 regionof SEQ ID NO:7 or a sequence with at least 85% similarity to SEQ IDNO:7, and a CDRH3 region of SEQ ID NO:8 or a sequence with at least 85%similarity to SEQ ID NO:8; (b) a light chain variable domain comprisinga CDRL1 region of SEQ ID NO:10 or a sequence with at least 85%similarity to SEQ ID NO:10, a CDRL2 region of SEQ ID NO:11 or a sequencewith at least 85% similarity to SEQ ID NO:11, and a CDRL3 region of SEQID NO:12 or a sequence with at least 85% similarity to SEQ ID NO:12 anda heavy chain variable domain comprising a CDRH1 region of SEQ ID NO:14or a sequence with at least 85% similarity to SEQ ID NO:14, a CDRH2region of SEQ ID NO:15 or a sequence with at least 85% similarity to SEQID NO:15, and a CDRH3 region of SEQ ID NO:16 or a sequence with at least85% similarity to SEQ ID NO:16; (c) a light chain variable domaincomprising a CDRL1 region of SEQ ID NO:18 or a sequence with at least85% similarity to SEQ ID NO:18, a CDRL2 region of SEQ ID NO:19 or asequence with at least 85% similarity to SEQ ID NO:19, and a CDRL3region of SEQ ID NO:20 or a sequence with at least 85% similarity to SEQID NO:20 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:22 or a sequence with at least 85% similarity to SEQ ID NO:22,a CDRH2 region of SEQ ID NO:23 or a sequence with at least 85%similarity to SEQ ID NO:23, and a CDRH3 region of SEQ ID NO:24 or asequence with at least 85% similarity to SEQ ID NO:24; (d) a light chainvariable domain comprising a CDRL1 region of SEQ ID NO:26 or a sequencewith at least 85% similarity to SEQ ID NO:26, a CDRL2 region of SEQ IDNO:27 or a sequence with at least 85% similarity to SEQ ID NO:27, and aCDRL3 region of SEQ ID NO:28 or a sequence with at least 85% similarityto SEQ ID NO:28and a heavy chain variable domain comprising a CDRH1region of SEQ ID NO:30 or a sequence with at least 85% similarity to SEQID NO:30, a CDRH2 region of SEQ ID NO:31 or a sequence with at least 85%similarity to SEQ ID NO:31, and a CDRH3 region of SEQ ID NO:32 or asequence with at least 85% similarity to SEQ ID NO:32; or (e) a lightchain variable domain comprising a CDRL1 region of SEQ ID NO:34 or asequence with at least 85% similarity to SEQ ID NO:34, a CDRL2 region ofSEQ ID NO:35 or a sequence with at least 85% similarity to SEQ ID NO:35,and a CDRL3 region of SEQ ID NO:36 or a sequence with at least 85%similarity to SEQ ID NO:36 and a heavy chain variable domain comprisinga CDRH1 region of SEQ ID NO:38 or a sequence with at least 85%similarity to SEQ ID NO:38, a CDRH2 region of SEQ ID NO:39 or a sequencewith at least 85% similarity to SEQ ID NO:39, and a CDRH3 region of SEQID NO:40 or a sequence with at least 85% similarity to SEQ ID NO:40. 2.The isolated bispecific antibody of claim 1, wherein the anti-CD30antibody or antigen binding portion thereof comprises a heavy and alight chain selected from the group consisting of: (a) a light chaincomprising SEQ ID NO:1 or a sequence with at least 85% similarity to SEQID NO:1, and a heavy chain comprising SEQ ID NO:5 or a sequence with atleast 85% similarity to SEQ ID NO:5; (b) a light chain comprising SEQ IDNO:9 or a sequence with at least 85% similarity to SEQ ID NO:9, and aheavy chain comprising SEQ ID NO:13 or a sequence with at least 85%similarity to SEQ ID NO:13; (c) a light chain comprising SEQ ID NO:17 ora sequence with at least 85% similarity to SEQ ID NO:17, and a heavychain comprising SEQ ID NO:21 or a sequence with at least 85% similarityto SEQ ID NO:21; (d) a light chain comprising SEQ ID NO:25 or a sequencewith at least 85% similarity to SEQ ID NO:25, and a heavy chaincomprising SEQ ID NO:29 or a sequence with at least 85% similarity toSEQ ID NO:29; and (e) a light chain comprising SEQ ID NO:33 or asequence with at least 85% similarity to SEQ ID NO:33, and a heavy chaincomprising SEQ ID NO:37 or a sequence with at least 85% similarity toSEQ ID NO:37.
 3. The isolated bispecific antibody of claim 1, whereinthe anti-CD30 antibody or antigen binding portion thereof is directly orindirectly conjugated to the T cell surface antigen antibody or antigenbinding portion thereof.
 4. The isolated bispecific antibody of claim 3,wherein the anti-CD30 antibody or antigen binding portion thereof isdirectly conjugated to the T cell surface antigen antibody or antigenbinding portion thereof.
 5. The isolated bispecific antibody of claim 4,wherein the anti-CD30 antibody or antigen binding portion thereof isdirectly conjugated to the T cell surface antigen antibody or antigenbinding portion thereof via a linker.
 6. The isolated bispecificantibody of claim 1, wherein the anti-CD3 antibody is a monoclonalantibody.
 7. The isolated bispecific antibody of claim 6, wherein theanti-CD3 antibody is OKT3 made by the hybridoma with ATCC accessionnumber CRL
 8001. 8. A pharmaceutical composition comprising the isolatedbispecific antibody of claim 1 and a pharmaceutically acceptablecarrier.
 9. A method of treating a patient having a CD30+ cancer, themethod comprising (a) administering a therapeutically effective amountof the bispecific antibody of claim 1 to reduce or inhibit CD30+ cancergrowth.
 10. The method of claim 9, wherein the CD30+ cancer is Hodgkin'slymphoma or AML.
 11. The method of claim 9, wherein the method furthercomprises administering an effective amount of a cancer treatment.
 12. Amethod of inhibiting growth of a tumor cell expressing CD30, comprisingcontacting the tumor cell with an effective amount of the antibody orantigen binding fragment thereof of claim 1 such that the growth of thecell is inhibited.
 13. A method of enhancing a T cell-mediated immuneresponse against a CD30+ tumor cell, the method comprising administeringa therapeutically effective amount of the bispecific antibody of claim 1to increase the T cell-mediated immune response as compared to treatmentwithout the bispecific antibody.
 14. An isolated bispecific antibodycapable of binding human CD30 and to a NK cell surface antigen, thebispecific antibody comprising an anti-CD30 antibody or antigen bindingportion thereof and an anti-NK cell surface antigen antibody or antigenbinding portion thereof, wherein the anti-NK cell surface antigenantibody or antigen binding portion thereof is an anti-CD16 antibody,and wherein the anti-CD30 antibody or antigen binding portion thereofcomprises: (a) a light chain variable domain comprising a CDRL1 regionof SEQ ID NO:2, a CDRL2 region of SEQ ID NO:3, and a CDRL3 region of SEQID NO:4 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:6, a CDRH2 region of SEQ ID NO:7, and a CDRH3 region of SEQ IDNO:8; (b) a light chain variable domain comprising a CDRL1 region of SEQID NO:10, a CDRL2 region of SEQ ID NO:11, and a CDRL3 region of SEQ IDNO:12 and a heavy chain variable domain comprising a CDRH1 region of SEQID NO:14, a CDRH2 region of SEQ ID NO:15, and a CDRH3 region of SEQ IDNO:16; (c) a light chain variable domain comprising a CDRL1 region ofSEQ ID NO:18, a CDRL2 region of SEQ ID NO:19, and a CDRL3 region of SEQID NO:20 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:22, a CDRH2 region of SEQ ID NO:23, and a CDRH3 region of SEQID NO:24; (d) a light chain variable domain comprising a CDRL1 region ofSEQ ID NO:26, a CDRL2 region of SEQ ID NO:27, and a CDRL3 region of SEQID NO:28 and a heavy chain variable domain comprising a CDRH1 region ofSEQ ID NO:30, a CDRH2 region of SEQ ID NO:31, and a CDRH3 region of SEQID NO:32; or (e) a light chain variable domain comprising a CDRL1 regionof SEQ ID NO:34, a CDRL2 region of SEQ ID NO:35, and a CDRL3 region ofSEQ ID NO:36 and a heavy chain variable domain comprising a CDRH1 regionof SEQ ID NO:38, a CDRH2 region of SEQ ID NO:39, and a CDRH3 region ofSEQ ID NO:40.
 15. The isolated bispecific antibody of claim 14, whereinthe anti-CD30 antibody or antigen binding portion thereof comprises aheavy and a light chain selected from the group consisting of: (a) alight chain comprising SEQ ID NO:1 or a sequence with at least 85%similarity to SEQ ID NO:1, and a heavy chain comprising SEQ ID NO:5 or asequence with at least 85% similarity to SEQ ID NO:5; (b) a light chaincomprising SEQ ID NO:9 or a sequence with at least 85% similarity to SEQID NO:9, and a heavy chain comprising SEQ ID NO:13 or a sequence with atleast 85% similarity to SEQ ID NO:13; (c) a light chain comprising SEQID NO:17 or a sequence with at least 85% similarity to SEQ ID NO:17, anda heavy chain comprising SEQ ID NO:21 or a sequence with at least 85%similarity to SEQ ID NO:21; (d) a light chain comprising SEQ ID NO:25 ora sequence with at least 85% similarity to SEQ ID NO:25, and a heavychain comprising SEQ ID NO:29 or a sequence with at least 85% similarityto SEQ ID NO:29; and (e) a light chain comprising SEQ ID NO:33 or asequence with at least 85% similarity to SEQ ID NO:33, and a heavy chaincomprising SEQ ID NO:37 or a sequence with at least 85% similarity toSEQ ID NO:37.
 16. An isolated bispecific antibody capable of bindinghuman CD30 and to a surface receptor on T cells, the bispecific antibodycomprising an anti-CD30 antibody or antigen binding portion thereof andan anti-CD3 antibody or antigen binding portion thereof, wherein theanti-CD30 antibody or antigen binding portion thereof comprises: (a) alight chain variable domain comprising a CDRL1 region of SEQ ID NO:2 ora sequence with at least 85% similarity to SEQ ID NO:2, a CDRL2 regionof SEQ ID NO:3 or a sequence with at least 85% similarity to SEQ IDNO:3, and a CDRL3 region of SEQ ID NO:4 or a sequence with at least 85%similarity to SEQ ID NO:4 and a heavy chain variable domain comprising aCDRH1 region of SEQ ID NO:6 or a sequence with at least 85% similarityto SEQ ID NO:6, a CDRH2 region of SEQ ID NO:7 or a sequence with atleast 85% similarity to SEQ ID NO:7, and a CDRH3 region of SEQ ID NO:8or a sequence with at least 85% similarity to SEQ ID NO:8; (b) a lightchain variable domain comprising a CDRL1 region of SEQ ID NO:10 or asequence with at least 85% similarity to SEQ ID NO:10, a CDRL2 region ofSEQ ID NO:11 or a sequence with at least 85% similarity to SEQ ID NO:11,and a CDRL3 region of SEQ ID NO:12 or a sequence with at least 85%similarity to SEQ ID NO:12 and a heavy chain variable domain comprisinga CDRH1 region of SEQ ID NO:14 or a sequence with at least 85%similarity to SEQ ID NO:14, a CDRH2 region of SEQ ID NO:15 or a sequencewith at least 85% similarity to SEQ ID NO:15, and a CDRH3 region of SEQID NO:16 or a sequence with at least 85% similarity to SEQ ID NO:16; (c)a light chain variable domain comprising a CDRL1 region of SEQ ID NO:18or a sequence with at least 85% similarity to SEQ ID NO:18, a CDRL2region of SEQ ID NO:19 or a sequence with at least 85% similarity to SEQID NO:19, and a CDRL3 region of SEQ ID NO:20 or a sequence with at least85% similarity to SEQ ID NO:20 and a heavy chain variable domaincomprising a CDRH1 region of SEQ ID NO:22 or a sequence with at least85% similarity to SEQ ID NO:22, a CDRH2 region of SEQ ID NO:23 or asequence with at least 85% similarity to SEQ ID NO:23, and a CDRH3region of SEQ ID NO:24 or a sequence with at least 85% similarity to SEQID NO:24; (d) a light chain variable domain comprising a CDRL1 region ofSEQ ID NO:26 or a sequence with at least 85% similarity to SEQ ID NO:26,a CDRL2 region of SEQ ID NO:27 or a sequence with at least 85%similarity to SEQ ID NO:27, and a CDRL3 region of SEQ ID NO:28 or asequence with at least 85% similarity to SEQ ID NO:28and a heavy chainvariable domain comprising a CDRH1 region of SEQ ID NO:30 or a sequencewith at least 85% similarity to SEQ ID NO:30, a CDRH2 region of SEQ IDNO:31 or a sequence with at least 85% similarity to SEQ ID NO:31, and aCDRH3 region of SEQ ID NO:32 or a sequence with at least 85% similarityto SEQ ID NO:32; or (e) a light chain variable domain comprising a CDRL1region of SEQ ID NO:34 or a sequence with at least 85% similarity to SEQID NO:34, a CDRL2 region of SEQ ID NO:35 or a sequence with at least 85%similarity to SEQ ID NO:35, and a CDRL3 region of SEQ ID NO:36 or asequence with at least 85% similarity to SEQ ID NO:36 and a heavy chainvariable domain comprising a CDRH1 region of SEQ ID NO:38 or a sequencewith at least 85% similarity to SEQ ID NO:38, a CDRH2 region of SEQ IDNO:39 or a sequence with at least 85% similarity to SEQ ID NO:39, and aCDRH3 region of SEQ ID NO:40 or a sequence with at least 85% similarityto SEQ ID NO:40.
 17. A method of eliciting an immune response in asubject to a tumor antigen, the method comprising administering thebispecific antibody of claim 16 in an amount effective to elicit animmune response.